Anti-nuclear antibodies (ANA) are a hallmark of systemic autoimmunity and of lupus erythematosus (SLE) in particular. Due to their prevalence in disease and specificity for ubiquitous self-antigens that normally induce self-tolerance, they have been the subjects of extensive investigation into mechanisms that lead to breaches in B cell self-tolerance during autoimmunity. Defining the origin of such antibodies, however, has proved to be problematic. This is because of germline sequence polymorphisms in Ig V region genes and because of extensive somatic diversity generated by processes that assemble V region genes during B lymphopoiesis in the bone marrow and that mutate them in the periphery during immune responses. To determine the origin of ANA, we developed a unique mouse model of spontaneous SLE in which V region gene expression by autoreactive B cells could be clearly defined and in which all somatic mutations could be unequivocally identified. In B6.Nba2 Tdt-/- Igh Igk mice, the absence of terminal deoxynucleotidyl transferase (Tdt) enabled us to identify every somatic mutation, even in CDR3, where untemplated addition of nucleotides during V gene assembly would otherwise obscure them. By reverting somatic mutations to germline sequence in spontaneous ANA-producing clones, we found in preliminary studies that almost all of the ANA originated from nonautoreactive antecedent B cells that acquired their autoreactivity via the process of somatic hypermutation (SHM). This finding leads us to hypothesize that activation-induced cytidine deaminase (AID) plays a paramount role in generating ANA of systemic autoimmune disease. In this application, we propose to test this mutation-founder hypothesis and its implications. We will use AID-deficient mice to determine if it applies to systemic autoimmunity in other spontaneous models of SLE and to specific types of ANA. We will determine if the requirement for T cell help in SLE, reported by numerous investigators, is explained solely by its role in generating the mutant autoreactive B cells or whether T cell help is also required after the mutant clones arise. Germline revertant nonautoreactive antibodies will be used as probes to define natural immunogens that recruited precursors to the ANA-producing mutant clones. We will develop a new model of tolerance in germline ANA-specific B cells and will determine whether mutant ANA generated by AID promote the escape of germline ANA-producing B cells by obscuring or removing nuclear antigens that would otherwise render the B cells tolerant. Our preliminary studies provide us with unique tools and information to address these issues. Results of this project will provide basic information about the natural history of autoreactive B cells, which is essential if we are to understand, control and prevent systemic autoimmune disease. PUBLIC HEALTH RELEVANCE: B lymphocytes that produce autoantibodies directed against self-tissues are major participants in autoimmune diseases such as systemic lupus erythematosus (SLE), scleroderma, Sjogren's syndrome and thyroiditis. A major goal of research in autoimmunity is to understand how such cells evade self-tolerance checkpoints that normally silence them functionally or physically under physiological circumstances. Progress in this area has been hampered by a lack of basic information regarding where these disease-participating cells arise and how they gain autoreactivity in the first place. Conventional wisdom holds that they first express autoantibodies when they are born in the adult bone marrow. But our preliminary data indicate that the autoantibodies of pathological B cells are created later in secondary tissues by mutation in the genes that specify the antibodies. In this study, we propose to test this basic hypothesis, define the disease-associated autoantibodies to which it applies and test prior interpretations regarding escape of such cells in autoimmune disease. By pinpointing the origin of autoreactive B cells and defining requirements for their progression in disease, we will establish a framework of knowledge that is essential to defining molecular targets for therapy and strategies to control or prevent systemic autoimmune disease.